​Check cells under microscope

Whilst growing, cells should be checked EVERY day to make sure they are healthy and growing as expected. They should be mainly attached to the bottom of the flask, round and plump or elongated in shape and refracting light around their membrane. This indicates they are healthy.

Media should be pinky orange in color. Discard the cells and do not use for the assay if they are detaching in large numbers and/or look shriveled and grainy/dark in color.

Preparing cell suspension

All cell handling and media preparation should be carried out using aseptic technique in class II safety cabinet.

When the cells are 70-80% confluent they should still be in the log phase of growth and can be used for plating.

First warm the culture medium in 37°C water bath for at least 30 min.

When ready, carefully pour off media from one 175 cm2 flask of the required cells into a waste pot (containing laboratory disinfectant) taking care not to increase contamination risk with any drips.

Using cell scraper, gently scrape the cells off the bottom of the flask into the media. Some cell lines may require some trypsinization. Check all the cells have come off by inspecting the base of the flask before moving on.

Make sure the cell suspension to be counted is well mixed by either gentle agitation of the flask. A serological pipette may be used if required.

Before the cells have a chance to settle at all, take out about 1 mL of cell suspension using a serological pipette and place in an eppendorf tube.

Do cell count using hemocytometer.

Obtaining correct cell density

Use the following calculation to obtain the correct cell density. This should be around 4.5 x 105 cells/mL but will require optimization depending on the cell line.

N = DF45 x 104

Where N is the cell count per mLWhere DF is the calculated dilution factor

As the cells are in 100 mL media the next calculation is:

100 mL x dilution factor

This will give a value for the volume of media the cells should be in.

Plating out the cells

Once the cells are resuspended in the correct amount of culture media, make sure the cells are well mixed by using a cell scraper to scrape off any cells that may have settled in the flask while waiting for the count. Carefully pour some cell suspension into a petri dish ready for plating.

Using a multichannel pipette and 300 µL pipette tips, pipette 200 µL of cell suspension from the petri dish into each well of the required 96-well culture plates. These plates should be labeled with the assay number, plate number and date.

Make sure the cells are well mixed at all times by gentle agitation of the flask each time the petri dish is filled back up as required and mixing every now and again while pipeting.

Place cell culture plates into a 37°C CO2 incubator overnight before using for an assay.